Polypeptides having lipase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having lipase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 09/703,416filed on Oct. 31, 2000, now U.S. Pat. No. 6,432,898 which is fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having lipaseactivity and isolated nucleic acid sequences encoding the polypeptides.The invention also relates to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid sequences as well as methods forproducing and using the polypeptides.

2. Description of the Related Art

Lipases (EC 3.1.1.3) are enzymes that can hydrolyze triglycerides torelease fatty acid.

Detergents formulated with lipolytic enzymes are known to have improvedproperties for removing fatty stains. For example, LIPOLASE™ (NovoNordisk A/S, Bagsvaerd, Denmark), a microbial lipase obtained from thefungus Thermomyces lanuginosus (also called Humicola lanuginosa), hasbeen introduced into many commercial brands of detergent.

WO 98/26057 discloses a polypeptide having lipase and phospholipaseactivity (GenBank Acc. No. A85215) obtained from Fusarium oxysporum.

It is an object of the present invention to provide improvedpolypeptides having lipase activity and nucleic acid encoding thepolypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having lipaseactivity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 85%identity with amino acids 31 to 349 of SEQ ID NO:2;

(b) a polypeptide encoded by a nucleic acid sequence which hybridizesunder high stringency conditions with (i) nucleotides 1525 to 2530 ofSEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 1525 to2530 of SEQ ID NO:1, (iii) a subsequence of (i) or (ii) of at least 100nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);

(c) a variant of the polypeptide having an amino acid sequence of SEQ IDNO:2 comprising a substitution, deletion, and/or insertion of one ormore amino acids; and

(d) a fragment of (a) or (b) that has lipase activity.

The present invention also relates to isolated nucleic acid sequencesencoding the polypeptides and to nucleic acid constructs, vectors, andhost cells comprising the nucleic acid sequences as well as methods forproducing and using the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of a Fusarium venenatum lipase (SEQ ID NOS:1 and 2,respectively).

FIG. 2 shows a restriction map of pSheB1.

FIG. 3 show a restriction map of pEJG60.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Lipase Activity

The term “lipase activity” is defined herein as a triacylglycerolacylhydrolase activity which catalyzes the hydrolysis of atriacylglycerol to diacylglycerol and a fatty acid anion.

A substrate for lipase is prepared by emulsifying tributyrin (glycerintributyrate) using gum Arabic as emulsifier. The hydrolysis oftributyrin at 30° C. at pH 7 is followed in a pH-stat titrationexperiment. One unit of lipase activity (1 LU) equals the amount ofenzyme capable of releasing 1 μmol butyric acid/minute at the standardconditions. 1 KLU=1000 LU. For purposes of the present invention,however, lipase activity is determined by measuring the hydrolysis of 2mM p-nitrophenyl butyrate in 100 mM MOPS pH 7.5, 4 mM CaCl₂, 990 μl ofDMSO, 80 μl of 1% AOS at pH 7.5, 25° C. One unit of lipase activity isdefined as 1.0 μmole of p-nitro phenolate anion produced per minute at25° C., pH 7.5.

In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 31 to 349 of SEQ ID NO:2 (i.e., the maturepolypeptide) of at least about 85%, preferably at least about 90%, morepreferably at least about 95%, and most preferably at least about 97%,which have lipase activity (hereinafter “homologous polypeptides”). In apreferred embodiment, the homologous polypeptides have an amino acidsequence which differs by five amino acids, preferably by four aminoacids, more preferably by three amino acids, even more preferably by twoamino acids, and most preferably by one amino acid from amino acids 31to 349 of SEQ ID NO:2. For purposes of the present invention, the degreeof identity between two amino acid sequences is determined by theClustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=1,gap penalty=3, windows=5, and diagonals=5.

Preferably, the polypeptides of the present invention comprise the aminoacid sequence of SEQ ID NO:2 or an allelic variant thereof; or afragment thereof that has lipase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 31 to 349 ofSEQ ID NO:2, or an allelic variant thereof; or a fragment thereof thathas lipase activity. In another preferred embodiment, the polypeptide ofthe present invention comprises amino acids 31 to 349 of SEQ ID NO:2. Inanother preferred embodiment, the polypeptide of the present inventionconsists of the amino acid sequence of SEQ ID NO:2 or an allelic variantthereof; or a fragment thereof that has lipase activity. In anotherpreferred embodiment, the polypeptide of the present invention consistsof the amino acid sequence of SEQ ID NO:2. In another preferredembodiment, the polypeptide consists of amino acids 31 to 349 of SEQ IDNO:2 or an allelic variant thereof; or a fragment thereof that haslipase activity. In another preferred embodiment, the polypeptideconsists of amino acids 31 to 349 of SEQ ID NO:2.

A fragment of SEQ ID NO:2 is a polypeptide having one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. Preferably, a fragment contains at least 260 amino acidresidues, more preferably at least 280 amino acid residues, and mostpreferably at least 300 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

In a second embodiment, the present invention relates to isolatedpolypeptides having lipase activity which are encoded by nucleic acidsequences which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with a nucleic acid probe which hybridizes underthe same conditions with (i) nucleotides 1525 to 2530 of SEQ ID NO:1,(ii) the cDNA sequence contained in nucleotides 1525 to 2530 of SEQ IDNO:1, (iii) a subsequence of (i) or (ii), or (iv) a complementary strandof (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.). The subsequence of SEQ ID NO:1 may be at least 100nucleotides or preferably at least 200 nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has lipase activity.The polypeptides may also be allelic variants or fragments of thepolypeptides that have lipase activity.

The nucleic acid sequence of SEQ ID NO:1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO:2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having lipase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicor cDNA of the genus or species of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, preferably at least 25, and morepreferably at least 35 nucleotides in length. Longer probes can also beused. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand, which encodes a polypeptide having lipase activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO:1 ora subsequence thereof, the carrier material is used in a Southern blot.For purposes of the present invention, hybridization indicates that thenucleic acid sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleic acid sequence shown in SEQ ID NO:1, itscomplementary strand, or a subsequence thereof, under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO:2, or a subsequencethereof. In another preferred embodiment, the nucleic acid probe is SEQID NO:1. In another preferred embodiment, the nucleic acid probe is themature polypeptide coding region of SEQ ID NO:1. In another preferredembodiment, the nucleic acid probe is the nucleic acid sequencecontained in plasmid pEJG60 which is contained in Escherichia coli NRRLB-30333, wherein the nucleic acid sequence encodes a polypeptide havinglipase activity. In another preferred embodiment, the nucleic acid probeis the mature polypeptide coding region contained in plasmid pEJG60which is contained in Escherichia coli NRRL B-30333.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third embodiment, the present invention relates to variants of thepolypeptide having an amino acid sequence of SEQ ID NO:2 comprising asubstitution, deletion, and/or insertion of one or more amino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof byan insertion or deletion of one or more amino acid residues and/or thesubstitution of one or more amino acid residues by different amino acidresidues. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

In a fourth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ IDNO:2 or the mature polypeptide thereof. The immunochemical propertiesare determined by immunological cross-reaction identity tests by thewell-known Ouchterlony double immunodiffusion procedure. Specifically,an antiserum containing polyclonal antibodies which are immunoreactiveor bind to epitopes of the polypeptide having the amino acid sequence ofSEQ ID NO:2 or the mature polypeptide thereof are prepared by immunizingrabbits (or other rodents) according to the, procedure described byHarboe and Ingild, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, AManual of Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 23, or Johnstone and Thorpe, Immunochemistryin Practice, Blackwell Scientific Publications, 1982 (more specificallypages 27-31). A polypeptide having immunochemical identity is apolypeptide which reacts with the antiserum in an identical fashion suchas total fusion of precipitates, identical precipitate morphology,and/or identical electrophoretic mobility using a specificimmunochemical technique. A further explanation of immunochemicalidentity is described by Axelsen, Bock, and Krøll, In N. H. Axelsen, J.Krøll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter10. A polypeptide having partial immunochemical identity is apolypeptide which reacts with the antiserum in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

The antibody may also be a monoclonal antibody. Monoclonal antibodiesmay be prepared and used, e.g., according to the methods of E. Harlowand D. Lane, editors, 1988, Antibodies, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 60%, even more preferably atleast 80%, even more preferably at least 90%, and most preferably atleast 100% of the lipase activity of the mature polypeptide of SEQ IDNO:2.

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may be a fungal polypeptide, andmore preferably a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ormore preferably a filamentous fungal polypeptide such as an Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

In another preferred embodiment, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

In another preferred embodiment, the polypeptide is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum polypeptide.

In a more preferred embodiment, the Fusarium venenatum cell is Fusariumvenenatum A3/5, which was originally deposited as Fusarium graminearumATCC 20334 and recently reclassified as Fusarium venenatum by Yoder andChristianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnellet al., 1998, Fungal Genetics and Biology 23: 57-67; as well astaxonomic equivalents of Fusarium venenatum regardless of the speciesname by which they are currently known. In another preferred embodiment,the Fusarium venenatum cell is a morphological mutant of Fusariumvenenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO97/26330.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. For example, taxonomic equivalentsof Fusarium are defined by D. L. Hawksworth, P. M. Kirk, B. C. Sutton,and D. N. Pegler (editors), 1995, In Ainsworth & Bisby's Dictionary ofthe Fungi, Eighth Edition, CAB International, University Press,Cambridge, England, pp.173-174.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other non-lipase polypeptides, e.g., at least about20% pure, preferably at least about 40% pure, more preferably about 60%pure, even more preferably about 80% pure, most preferably about 90%pure, and even most preferably about 95% pure, as determined bySDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequenceswhich encode a polypeptide of the present invention. In a preferredembodiment, the nucleic acid sequence is set forth in SEQ ID NO:1. Inanother more preferred embodiment, the nucleic acid sequence is thesequence contained in plasmid pEJG60 that is contained in Escherichiacoli NRRL B-30333. In another preferred embodiment, the nucleic acidsequence is the mature polypeptide coding region of SEQ ID NO:1. Inanother more preferred embodiment, the nucleic acid sequence is themature polypeptide coding region contained in plasmid pEJG60 that iscontained in Escherichia coli NRRL B-30333. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof,which differ from SEQ ID NO:1 by virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO:1which encode fragments of SEQ ID NO:2 that have lipase activity.

A subsequence of SEQ ID NO:1 is a nucleic acid sequence encompassed bySEQ ID NO:1 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 780nucleotides, more preferably at least 840 nucleotides, and mostpreferably at least 900 nucleotides. The present invention also relatesto mutant nucleic acid sequences comprising at least one mutation in themature polypeptide coding sequence of SEQ ID NO:1, in which the mutantnucleic acid sequence encodes a polypeptide which consists of aminoacids 31 to 349 of SEQ ID NO:2.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Fusarium, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, CDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

The present invention also relates to nucleic acid sequences which havea degree of homology to the mature polypeptide coding sequence of SEQ IDNO:1 (i.e., nucleotides 1525 to 2530) of at least about 85%, preferablyabout 90%, more preferably about 95%, and most preferably about 97%homology, which encode an active polypeptide. For purposes of thepresent invention, the degree of homology between two nucleic acidsequences is determined by the Wilbur-Lipman method (Wilbur and Lipman,1983, Proceedings of the National Academy of Science USA 80: 726-730)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=3, gap penalty=3, and windows=20.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor lipase activity to identify amino acid residues that are critical tothe activity of the molecule. Sites of substrate-enzyme interaction canalso be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize undervery low stringency conditions, preferably low stringency conditions,more preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with anucleic acid probe which hybridizes under the same conditions with thenucleic acid sequence of SEQ ID NO:1 or its complementary strand; orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) the cDNA sequencecontained in nucleotides 1525 to 2530 of SEQ ID NO:1, (iii) asubsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii),or (iii); and (b) isolating the nucleic acid sequence. The subsequenceis preferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has lipase activity.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe mature polypeptide coding sequence of SEQ ID NO:1 or a subsequencethereof, wherein the mutant nucleic acid sequence encodes a polypeptidewhich consists of amino acids 31 to 349 of SEQ ID NO:2 or a fragmentthereof which has lipase activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” is defined herein as anucleic acid sequence which directly specifies the amino acid sequenceof its protein product. The boundaries of a genomic coding sequence aregenerally determined by a ribosome binding site (prokaryotes) or by theATG start codon (eukaryotes) located just upstream of the open readingframe at the 5′ end of the mRNA and a transcription terminator sequencelocated just downstream of the open reading frame at the 3′ end of themRNA. A coding sequence can include, but is not limited to, DNA, cDNA,and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xyIA and xyIBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

In a preferred embodiment, the signal peptide coding region isnucleotides 1376 to 1420 of SEQ ID NO:1 which encode amino acids 1 to 15of SEQ ID NO:2.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred embodiment, the propeptide coding region is nucleotides1421 to 1465 of SEQ ID NO:1 which encode amino acids 16 to 30 of SEQ IDNO:2.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more targeting sequences canbe used. For example, a circular plasmid or DNA fragment preferablyemploys a single targeting sequence, while a linear plasmid or DNAfragment preferably employs two targeting sequences.

The regulatory sequence of the construct can be comprised of one or morepromoters, enhancers, scaffold-attachment regions or matrix attachmentsites, negative regulatory elements, transcription binding sites, orcombinations of these sequences.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′ non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon. Typically, the first exon lies 5′ of the second exon,and the splice-donor site overlapping and flanking the first exon on its3′ side recognizes a splice-acceptor site flanking the second exon onthe 5′ side of the second exon. A splice-acceptor site, like asplice-donor site, is a sequence which directs the splicing of one exonto another exon. Acting in conjunction with a splice-donor site, thesplicing apparatus uses a splice-acceptor site to effect the removal ofan intron.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid, sequence of the present invention maybe expressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of, which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for integration of the vector into the genome by homologousor nonhomologous recombination. Alternatively, the vector may containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are generally characterizedby a mycelial wall composed of chitin, cellulose, glucan, chitosan,mannan, and other complex polysaccharides. Vegetative growth is byhyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred embodiment, the filamentous fungal host cellis a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce the polypeptide; and (b) recovering thepolypeptide. Preferably, the strain is of the genus Fusarium, and morepreferably Fusarium venenatum.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the mature polypeptide coding region of SEQ IDNO:1, wherein the mutant nucleic acid sequence encodes a polypeptidewhich consists of amino acids 31 to 349 of SEQ ID NO:2, and (b)recovering the polypeptide.

The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulatory sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleic acid sequenceencoding a polypeptide having lipase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and Theologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as festuca, lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu etal.,1993, Plant Molecular Biology 22: 573-588).

A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having lipase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Lipase Activity

The present invention also relates to methods for producing a mutantcell of a parent cell, which comprises disrupting or deleting a nucleicacid sequence encoding the polypeptide or a control sequence thereof,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The construction of strains which have reduced lipase activity may beconveniently accomplished by modification or inactivation of a nucleicacid sequence necessary for expression of the polypeptide having lipaseactivity in the cell. The nucleic acid sequence to be modified orinactivated may be, for example, a nucleic acid sequence encoding thepolypeptide or a part thereof essential for exhibiting lipase activity,or the nucleic acid sequence may have a regulatory function required forthe expression of the polypeptide from the coding sequence of thenucleic acid sequence. An example of such a regulatory or controlsequence may be a promoter sequence or a functional part thereof, i.e.,a part which is sufficient for affecting expression of the polypeptide.Other control sequences for possible modification are described above.

Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting orscreening for cells in which the lipase producing capability has beenreduced. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced lipase activity or production.

Modification or inactivation of production of a polypeptide of thepresent invention may be accomplished by introduction, substitution, orremoval of one or more nucleotides in the nucleic acid sequence encodingthe polypeptide or a regulatory element required for the transcriptionor translation thereof. For example, nucleotides may be inserted orremoved so as to result in the introduction of a stop codon, the removalof the start codon, or a change of the open reading frame. Suchmodification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleicacid sequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce production by ahost cell of choice is by gene replacement or gene interruption. In thegene interruption method, a nucleic acid sequence corresponding to theendogenous gene or gene fragment of interest is mutagenized in vitro toproduce a defective nucleic acid sequence which is then transformed intothe host cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous gene or genefragment. It may be desirable that the defective gene or gene fragmentalso encodes a marker which may be used for selection of transformantsin which the gene encoding the polypeptide has been modified ordestroyed.

Alternatively, modification or inactivation of the nucleic acid sequencemay be performed by established anti-sense techniques using a nucleotidesequence complementary to the polypeptide encoding sequence. Morespecifically, production of the polypeptide by a cell may be reduced oreliminated by introducing a nucleotide sequence complementary to thenucleic acid sequence encoding the polypeptide which may be transcribedin the cell and is capable of hybridizing to the polypeptide mRNAproduced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the polypeptide mRNA, theamount of polypeptide translated is thus reduced or eliminated.

It is preferred that the cell to be modified in accordance with themethods of the present invention is of microbial origin, for example, afungal strain which is suitable for the production of desired proteinproducts, either homologous or heterologous to the cell.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleic acid sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide than the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity byfermentation of a cell which produces both a polypeptide of the presentinvention as well as the protein product of interest by adding aneffective amount of an agent capable of inhibiting lipase activity tothe fermentation broth before, during, or after the fermentation hasbeen completed, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity bycultivating the cell under conditions permitting the expression of theproduct, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the lipase activity substantially,and recovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with a lipase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the lipase activity. Complete removal of lipase activity may beobtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 6.5-7 and a temperature in the range of 25-40° C. fora sufficient period of time to attain the desired effect, wheretypically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallylipase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thelipase-deficient cells may also be used to express heterologous proteinsof pharmaceutical interest such as hormones, growth factors, receptors,and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from lipase activity which is produced by a method ofthe present invention.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention. Preferably, thecompositions are enriched in a polypeptide of the present invention. Inthe present context, the term “enriched” indicates that the lipaseactivity of the composition has been increased, e.g., with an enrichmentfactor of 1.1.

The composition may comprise a polypeptide of the invention as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase. The additionalenzyme(s) may be producible by means of a microorganism belonging to thegenus Aspergillus, preferably Aspergillus aculeatus, Aspergillusawamori, Aspergillus niger, or Aspergillus oryzae, or Trichoderma,Humicola, preferably Humicola insolens, or Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having lipase activity in any industrial application oflipases, e.g., in detergents.

Use in Detergent

The variant may be used as a detergent additive, e.g., at aconcentration (expressed as pure enzyme protein) of 0.001-10 (e.g.,0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg perliter of wash liquor.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations. In a laundry detergent, the variant may be effective for theremoval of fatty stains, for whiteness maintenance and for dingycleanup. A laundry detergent composition may be formulated as describedin WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939 and WO97/43375.

The detergent composition of the invention may particularly beformulated for hand or machine dishwashing operations e.g., as describedin GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble). In adishwashing composition, the variant may be effective for removal ofgreasy/oily stains, for prevention of the staining/discoloration of thedishware and plastic components of the dishwasher by highly coloredcomponents and the avoidance of lime soap deposits on the dishware.

Use in Degumming

A polypeptide of the present invention may be used for degumming anaqueous carbohydrate solution or slurry to improve its filterability,particularly, a starch hydrolysate, especially a wheat starchhydrolysate which is difficult to filter and yields cloudy filtrates.The treatment may be performed using methods well known in the art. See,for example, EP 219,269 and EP 808,903.

Signal Peptide and Propeptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to one or both of a firstnucleic acid sequence consisting of nucleotides 1376 to 1429 of SEQ IDNO:1 encoding a signal peptide consisting of amino acids 1 to 15 of SEQID NO:2 and a second nucleic acid sequence consisting of nucleotides1421 to 1465 of SEQ ID NO:1 encoding a propeptide consisting of aminoacids 16 to 30 of SEQ ID NO:2, wherein the gene is foreign to the firstand second nucleic acid sequences.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The first and second nucleic acid sequences may be operably linked toforeign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As noted earlier, where both signal peptide-andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred embodiment, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred embodiment, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Fusarium venenatum WTY700 3.8d, a spore-purified tri5-minus, dps1-minusstrain, was used as the recipient strain for transformation experiments.Fusarium venenatum WTY700 3.8d is a morphological mutant of Fusariumvenenatum strain ATCC 20334 (Wiebe et al., 1991, Mycol. Research 95:1284-1288),

Example 1

Fermentation and Mycelial Tissue

Fusarium venenatum WTY700 3.8d was grown in a two-liter lab-scalefermentor using a fed-batch fermentation scheme with NUTRIOSE™ (RoquetteFreres, S. A., Beinheim, France) as the carbon source and yeast extract.Ammonium phosphate was provided in the feed. The fermentation wasmaintained at pH 6-6.5 and 30 ° C. with positive dissolved oxygen.

Mycelial samples were harvested at 2, 4, 6, and 8 days post-inoculum andquick-frozen in liquid nitrogen. The samples were stored at −80° C.until they were disrupted for RNA extraction.

Example 2

cDNA Library Construction

Total cellular RNA was extracted from the mycelial samples described inExample 1 according to the method of Timberlake and Barnard (1981, Cell26: 29-37), and the RNA samples were analyzed by Northern hybridizationafter blotting from 1% formaldehyde-agarose gels (Davis et al., 1986,Basic Methods in Molecular Biology, Elsevier Science Publishing Co.,Inc., New York). Polyadenylated mRNA fractions were isolated from totalRNA with an mRNA Separator Kit™ (Clontech Laboratories, Inc., Palo Alto,Calif.) according to the manufacturer's instructions. Double-strandedcDNA was synthesized, using approximately 5 μg of poly(A)+mRNA accordingto the method of Gubler and Hoffman (1983, Gene 25: 263-269) except aNotI-(dT)18 primer (Pharmacia Biotech, Inc., Piscataway, N.J.) was usedto initiate first strand synthesis. The cDNA was treated with mung beannuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and theends were made blunt with T4 DNA polymerase (New England Biolabs,Beverly, Mass.).

The cDNA was digested with NotI, size selected by agarose gelelectrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (InvitrogenCorporation, Carlsbad, Calif.) which had been cleaved with NotI plusEcoRV and dephosphorylated with calf-intestine alkaline phosphatase(Boehringer Mannheim Corporation, Indianapolis, Ind.). The ligationmixture was used to transform competent E. coli TOP10 cells (InvitrogenCorporation, Carlsbad, Calif.). Transformants were selected on 2YT agarplates (Miller, 1992, A Short Course in Bacterial Genetics. A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.) which contained kanamycinat a final concentration of 50 μg/ml.

Example 3

Template Preparation and Nucleotide Sequencing

From the cDNA library described in Example 2, 1192 transformant colonieswere picked directly from the transformation plates into 96-wellmicrotiter dishes which contained 200 μl of 2YT broth (Miller, 1992,supra) with 50 μg/ml kanamycin. The plates were incubated overnight at37° C. without shaking. After incubation 100 μl of sterile 50% glycerolwas added to each well. The transformants were replicated intosecondary, deep-dish 96-well microculture plates (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) containing 1 ml ofMagnificent Broth™ (MacConnell Research, San Diego, Calif.) supplementedwith 50 μg of kanamycin per ml in each well. The primary microtiterplates were stored frozen at −80° C. The secondary deep-dish plates wereincubated at 37° C. overnight with vigorous agitation (300 rpm) onrotary shaker. To prevent spilling and cross-contamination, and to allowsufficient aeration, each secondary culture plate was covered with apolypropylene pad (Advanced Genetic Technologies Corporation,Gaithersburg, Md.) and a plastic microtiter dish cover.

DNA was isolated from each well using the 96-well Miniprep Kit protocolof Advanced Genetic Technologies Corporation (Gaithersburg, Md.) asmodified by Utterback et al. (1995, Genome Sci. Technol. 1: 1-8).Single-pass DNA sequencing (EST) was done with a Perkin-Elmer AppliedBiosystems Model 377 XL Automated DNA Sequencer (Perkin-Elmer/AppliedBiosystems, Inc., Foster City, Calif.) using dye-terminator chemistry(Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and thereverse lac sequencing primer.

Example 4

Analysis of DNA Sequence Data

Nucleotide sequence data were scrutinized for quality, and samplesgiving improper spacing or ambiguity levels exceeding 3% were discardedor re-run. Vector sequences and ambiguous base calls at the ends of theDNA sequences were trimmed with assistance of FACTURA™ software(Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif. Allsequences were compared to each other to determine multiplicity usingAutoAssembler™ software (Perkin-Elmer Applied Biosystems, Inc., FosterCity, Calif.). Lastly, all sequences were translated in three frames andsearched against a non-redundant database (NRDB) using GeneAssist™software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.)with a modified Smith-Waterman algorithm using the BLOSUM 62 matrix witha threshold score of 70. The NRDB was assembled from Genpept,Swiss-Prot, and PIR databases.

Example 5

Identification of Lipase 1 cDNA Clones

Putative lipase clones were identified by comparing the deduced aminoacid sequence of the ESTs to protein sequences deposited in publiclyavailable databases such as Swissprot, Genpept, and PIR using a modifiedSmith-Waterman search algorithm (Perkin-Elmer Applied Biosystems, FosterCity, Calif.). Tentative identification was based on amino acid sequencesimilarity to numerous fungal lipases. One clone, Fusarium venenatum ESTFA0726, was selected for nucleotide sequence analysis which revealedthat the cDNA clone was truncated at its 5 prime end.

Example 6

Fusarium Venenatum Genomic DNA Extraction

Fusarium venenatum WTY700 was grown for 24 hours at 28° C. and 150 rpmin 25 ml of YEG medium composed per liter of 5 g of yeast extract and 20g of glucose. Mycelia were then collected by filtration throughMiracloth (Calbiochem, La Jolla, Calif.) and washed once with 25 ml of10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer was drained from themycelia which were subsequently frozen in liquid nitrogen. The frozenmycelia were ground to a fine powder in an electric coffee grinder, andthe powder was added to 20 ml of TE buffer and 5 ml of 20% w/v sodiumdodecylsulfate (SDS) in a disposable plastic centrifuge tube. Themixture was gently inverted several times to ensure mixing, andextracted twice with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to givea final concentration of 0.3 M and the nucleic acids were precipitatedwith 2.5 volumes of ice cold ethanol. The tube was centrifuged at15,000×g for 30 minutes and the pellet was allowed to air dry for 30minutes before resuspension in 0.5 ml of TE buffer. DNase-freeribonuclease A was added to a concentration of 100 μg/ml and the mixturewas incubated at 37° C. for 30 minutes. Proteinase K (200 μg/ml) wasthen added and the mixture was incubated an additional hour at 37° C.Finally, the mixture was extracted twice with phenol:chloroform:isoamylalcohol (25:24:1 v/v/v) before precipitating the DNA with sodium acetateand ethanol according to standard procedures. The DNA pellet was driedunder vacuum, resuspended in TE buffer, and stored at 4° C.

Example 7

Genomic DNA Library Construction, Screening, and Isolation of GenomicLipase 1 Clone

Genomic libraries of Fusarium venenatum WTY700 were constructed inλZipLox according to the manufacturer's instructions (Life Technologies,Gaithersburg, Md.). Fusarium venenatum genomic DNA was partiallydigested with Tsp5091 and size-fractionated on 1% agarose gels. DNAfragments migrating in the size range 3-7 kb were excised and elutedfrom the agarose gel slices using Prep-a-Gene reagents (BioRad,Hercules, Calif.). The eluted DNA fragments were ligated withEcoRI-cleaved and dephosphorylated λZipLox vector arms (LifeTechnologies, Gaithersburg, Md.), and the ligation mixtures werepackaged using commercial packaging extracts (Stratagene, La Jolla,Calif.). The packaged DNA libraries were plated and amplified in E. coliY1090ZL cells.

The cDNA from Fusarium venenatum clone FA0726 was excised from thevector plasmid by digestion with EcoRI and NotI yielding anapproximately 900 bp fragment. The fragment was purified by gelelectrophoresis, and radiolabeled with α[³²P] dCTP using a Prime-itRandom Primer Labeling Kit (Stratagene, La Jolla, Calif.).

Approximately 40,000 plaques from the library were screened byplaque-hybridization (Davis et al., 1980, supra) with the radiolabeledprobe fragment of the Fusarium venenatum lipase gene using highstringency conditions at 45° C. (high stringency=50% formamide, 5×SSPE,0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA). Plaques,which gave hybridization signals, were purified once in E. coli DH10Bcells, and the individual clones were subsequently excised from theλZipLox vector as pZL1-derivatives (D'Alessio et al., 1992, Focus® 14:7).

One plaque was identified that hybridized strongly to the Fusariumvenenatum lipase gene probe, and was subsequently excised from theλZipLox vector as a pZL1-derivative (D'Alessio et al., 1992, supra).Plasmid DNA was isolated from the clone by passage through E. coli DH10Bcells (Life Technologies, Gaithersburg, Md.) according to themanufacturer's instructions. This clone was designated E. coliDH10B-pFvLipase1.

Example 8

Characterization of the Fusarium Venenatum Genomic Clone Encoding Lipase1

DNA sequencing was performed on an Perkin-Elmer Biosystems Model 377 XLAutomated DNA Sequencer using dye-terminator chemistry (Giesecke et al.,1992, Journal of Virology Methods 38: 47-60). Contig sequences weregenerated using a transposon insertion strategy (Primer IslandTransposition Kit, Perkin-Elmer/Applied Biosystems, Inc., Foster City,Calif.). The 2.94 kb genomic fragment was sequenced to an averageredundancy of 4.8.

The nucleotide sequence and deduced amino acid sequence are shown inFIG. 1. The insert contains an open reading frame of 1.153 kb encoding apolypeptide of 350 amino acids. Using the SignalP software program(Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal peptide of15 residues was predicted. The predicted signal peptide is followed by a15 residue propeptide ending with a concanical propeptide Glu/Argcleavage site. N-terminal sequencing of the lipase 1 protein supportsthis propeptide cleavage site prediction. The open reading frame isinterrupted by two introns of 49 bp and 58 bp. Thus, the mature Fusariumvenenatum lipase comprises 319 amino acids and a predicted molecularweight of 33.6 kDa. There are 2 potential N-linked glycosylation sites(Asn-X-Ser/Thr) within Fusarium venenatum lipase 1.

A comparative alignment of lipase sequences using the Clustal Walgorithm in the Megalign program of DNA-Star, showed that the deducedamino acid sequence of the Fusarium venenatum lipase 1 gene shares 81%identity to the deduced amino acid sequence of a Fusarium oxysporumphospholipase A (EP0869167).

Example 9

Construction of Plasmid pSheB1

The Fusarium venenatum expression vector pSheB1 (FIG. 2) was generatedby modification of pDM181 (WO 98/20136). The modifications included (a)removal of two NcoI sites within the pDM181 sequence, and (b)restoration of the natural translation start of the Fusarium oxysporumtrypsin promoter (reconstruction of an NcoI site at the ATG startcodon).

Removal of two NcoI sites within the pDM181 sequence was accomplishedusing the QuikChange™ site-directed mutagenesis kit (Stratagene CloningSystems, La Jolla, Calif.) according to the manufacturer's instructionwith the following pairs of mutagenesis primers:

5′-dCAGTGAATTGGCCTCGATGGCCGCGGCCGCGAATT-3′ plus (SEQ ID NO:3)5′-dAATTCGCGGCCGCGGCCATCGAGGCCAATTCACTG-3′ (SEQ ID NO:4)5′-dCACGAAGGAAAGACGATGGCTTTCACGGTGTCTG-3′ plus (SEQ ID NO:5)5′-dCAGACACCGTGAAAGCCATCGTCTTTCCTTCGTG-3′ (SEQ ID NO:6)

Restoration of the natural translation start of the Fusarium oxysporumtrypsin promoter was also accomplished using the Stratagene QuikChange™site directed mutagenesis kit in conjunction with the following pair ofmutagenesis primers:

5′-dCTATCTCTTCACCATGGTACCTTAATTAAATACCTTGTTGGAAGCG-3′ plus (SEQ ID NO:7)5′-dCGCTTCCAACAAGGTATTTAATTAAGGTACCATGGTGAAGAGATAG-3′ (SEQ ID NO:8)

All site-directed changes were confirmed by DNA sequence analysis of theappropriate vector regions.

Example 10

Construction of Expression Vector pEJG60

The lipase-expression vector, pEJG60 was constructed as follows. Thelipase coding region was amplified from pFvlipase1 using the followingpair of primers:

Primer 990658: 5′-CGTTCTTTGTCTGTCAGCATGCATCTCCTATCACTCC-3′ (SEQ ID NO:9)Primer 990661: 5′-CCAGAGTTTTTGTTATGGTTAATTAATATCGTTACTGCGTAAATG-3′ (SEQID NO:10)

The forward primer introduces a SphI site which contains the ATG, andthe reverse primer introduces a PacI site after the stop codon.

The amplification reaction (100 μl) contained the following components:0.5 μg of genomic clone, pFvLipase1, 50 pmol of the forward primer, 50pmol of the reverse primer, 10 mM dNTPs (dATP, dCTP, dGTP, and dTTP),1×Pwo DNA polymerase buffer, and 2.5 units of Pwo DNA polymerase(Boehringer Mannheim, Indianapolis, Ind.). The reactions were incubatedin a Perkin-Elmer Model 480 Thermal Cycler programmed for 1 cycles at95° C. for 2 minutes; 10 cycles at 94° C. for 45 seconds, 55° C. for 45seconds, and 72° C. for 2 minutes; 17 cycles at 94° C. for 45 seconds,55° C. for 45 seconds, and 72° C. for 2 minutes with an extension of 20seconds per cycle; 1 cycle at 72° C. for 10 minutes; and a soak cycle at4° C. The reaction products were isolated on a 1% agarose gel where a1.15 kb product band was excised from the gel and purified using QiaquikGel Extraction Kit (Qiagen, Chatsworth, Calif.) according to themanufacturer's instructions.

The generated fragment was digested with SphI, blunted with Klenow,digested with PacI, and purified by agarose gel electrophoresis andQiaquik Gel Extraction Kit (Qiagen, Chatsworth, Calif.). The purifiedDNA segment was ligated into pSheB1 (FIG. 2) which was previously NcoIdigested, treated with DNA polymerase I (Klenow fragment), and digestedwith PacI. The treatment of the NcoI-digested vector with Klenowfragment resulted in a filling in of the NcoI cohesive end, therebymaking it blunt and compatible with the blunt site of the lipase DNAsegment. The resulting expression plasmid was designated pEJG60 (FIG.3). The PCR-amplified lipase gene segment was re-sequenced to verify theabsence of any errors.

Example 10

Transformation of Fusarium Venenatum and Analysis of Fusarium VenenatumTransformants

Spores of Fusarium venenatum WTY700 were generated by inoculating aflask containing 500 ml of RA sporulation medium with 10 plugs from a1×Vogels medium plate (2.5% Noble agar) supplemented with 2.5% glucoseand 2.5 mM sodium nitrate and incubating at 28° C., 150 rpm for 2 to 3days. Spores were harvested through Miracloth (Calbiochem, San Diego,Calif.) and centrifuged 20 minutes at 7000 rpm in a Sorvall RC-5Bcentrifuge (E. I. DuPont De Nemours and Co., Wilmington, Del.). Pelletedspores were washed twice with sterile distilled water, resuspended in asmall volume of water, and then counted using a hemocytometer.

Protoplasts were prepared by inoculating 100 ml of YEPG medium with4×10⁷ spores of Fusarium venenatum WTY700 and incubating for 16 hours at24° C. and 150 rpm. The culture was centrifuged for 7 minutes at 3500rpm in a Sorvall RT 6000D (E. I. DuPont De Nemours and Co., Wilmington,Del.). Pellets were washed twice with 30 ml of 1 M MgSO₄ and resuspendedin 15 ml of 5 mg/ml of NOVOZYME 234™ (batch PPM 4356, Novo Nordisk A/S,Bagsvaerd, Denmark) in 1 M MgSO₄. Cultures were incubated at 24° C. and150 rpm until protoplasts formed. A volume of 35 ml of 2 M sorbitol wasadded to the protoplast digest and the mixture was centrifuged at 2500rpm for 10 minutes. The pellet was resuspended, washed twice with STC,and centrifuged at 2000 rpm for 10 minutes to pellet the protoplasts.Protoplasts were counted with a hemocytometer and resuspended in an8:2:0.1 solution of STC:SPTC:DMSO to a final concentration of 1.25×10⁷protoplasts/ml. The protoplasts were stored at −80° C., aftercontrolled-rate freezing in a Nalgene Cryo 1° C. Freezing Container (VWRScientific, Inc., San Francisco, Calif.).

Frozen protoplasts of Fusarium venenatum WTY700 were thawed on ice. Fiveμg of pEJG60 described in Example 10 and 5 μl of heparin (5 mg per ml ofSTC) was added to a 50 ml sterile polypropylene tube. One hundred μl ofprotoplasts was added, mixed gently, and incubated on ice for 30minutes. One ml of SPTC was added and incubated 20 minutes at roomtemperature. After the addition of 25 ml of 40° C. COVE top agarose, themixture was poured onto an empty 150 mm diameter plate and incubatedovernight at room temperature. Then an additional 25 ml of 40° C. COVEtop agarose containing 10 mg of BASTA™ per ml was poured on top of theplate and incubated at room temperature for up to 14 days. The activeingredient in the herbicide BASTA™ is phosphinothricin. BASTA™ wasobtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and wasextracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), andonce with chloroform:isoamyl alcohol (24:1) before use.

Twenty-four transformants were picked directly from the selection plates(COVE underlay with COVE-BASTA™ overlay) and inoculated into 125 mlshake flasks containing 25 ml of M400Da medium supplemented with 1 mMCaCl₂ and 100 μg/ml ampicillin (to prevent bacterial contamination) andincubated at 28° C., 200 rpm on a platform shaker for 7 days. Theuntransformed recipient strain was also included as a negative control.

Flasks were sampled at 5 and 7 days and assayed for lipase activity asdescribed below. The samples were also submitted to SDS-PAGE using Novexgradient gels (Novex Experimental Technology, San Diego, Calif.).

Lipase activity was determined as follows: 100 μl of substrate (3.92 mlof 100 mM MOPS pH 7.5, 4 mM CaCl₂, 990 μl of DMSO, 80 μl of 1% AOS, and20 μl of p-nitrophenyl butyrate) was added to 100 μl of diluted sample.The samples were diluted accordingly in 100 mM MOPS pH 7.5, 4 mM CaCl₂.The absorbance at 405 nm was monitored for 3 minutes at room temperaturein a 96-well microtiter plate using a Molecular Devices ThermomaxMicroplate Reader.

The lipase assay results indicated that at both 5 and 7 days, most ofthe transformants produced lipase activity well above that of theuntransformed control. Shake flask culture broths from transformants #1and #3, the two highest scorers in the lipase assay, were analyzed on a16% tricine gel. A prominent polypeptide at a apparent molecular weightof 32-33 kD was observed at both time points and for each transformantharboring pEJG60.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coil pEJG60 NRRL B-30333August 22, 2000

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 10 <210> SEQ ID NO 1 <211> LENGTH: 2940<212> TYPE: DNA <213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 1aattcatgtg aatctactat gtaacagtat gttgtattgc attacccatc aa#cgttgaat     60cgttgcgacg taacggcccg gttcaagcga gatgtagata tgttggtagt ta#attgatgg    120gttaggtatt cttttcatca actcggtatt ctcattcccc agatatcggc ac#ttgtcttt    180actccagatt tcatatcgca tcgagttata tacagtccca attgagtcga ct#accccgtc    240caaaacaggt tttctcacaa accaaccgca gcctaacaaa aagtcccttg tc#tttctgca    300ataaatgctg acaccccctg gctttttagg actgacggct cacgatgcag cc#gttgcgat    360aattaattga caattacccg cacattgatg catacttggc ggtcaggtca gg#tcaggctg    420aagcatacct attgggtcat ttatttgccg atcgtggtga aaagaatgca ag#tgataact    480agttacgagt cgctttatga aagatggttg gtcgaaactg tcaatatggc at#gggcggca    540aatcgtttgg tctcaactct atagcatgta ctataattgg tcttttcatc ac#agtcacgc    600caaagtgcca gtctcagact atggaccaac cactttcctc cttcacgtct aa#attgactt    660gatcaccaga ctcgaatatt ttttcttttc ttctataccc ctaggatcat ac#aatacgaa    720ccccaactca actcgagaga gagagtcccc ttcccaacat tttgacagcc ct#tgctcttc    780tcctcccagg atgtaacaga agctgaaagg gtacccctgt agcccacctt ta#cccaccat    840cttttccatc tgtatcggtg catcccatca caaccctcac gtggtccgag at#cgtcgtta    900cccgtattgg aagctcactc cgggcccaac gagagattgg accaaggaaa aa#taactttg    960agacctcttc aagcagtcgg tcattcgtta ctgggatgtg tagtcgataa tg#cggggtga   1020caggccctca atccagcacc caccatcatg ggcactgact gtactaccgg ag#cccatcat   1080ttcgtttttg ggtcctggcg tctacttgac cgactgagtt tgccaagatg ga#tggcatga   1140gagacagtgg ttaggctggg cgggtattgt gatgagagaa agcgagagac ta#gttagaag   1200caaagaaaaa agatatataa gctgtcacat ccctcatgaa catgctgttc tt#gtaagtcg   1260ggatatcagg gccagcttca gtattcagta tcctttctga gggagttgca cc#ttgtcaca   1320gcttgtctgt ctatcactta tacttaccct tggaccacgt tctttgtctg tc#aagatgca   1380tctcctatca ctcctctcaa ttgccaccct tgcggtagcc agccctctga gc#gttgaaga   1440ttacgccaag gctctcgatg aaagaggtaa aacgattctc tgttcccata ac#aattccaa   1500tactcacaga cctagctgtt tctgtctcta ccaacgactt tggcaacttc aa#gttctaca   1560tccagcacgg tgccgcagca tactgtaact ctgaagccgc agccggtgca aa#ggtcacct   1620gcggaggaaa cggttgccca acggtccagt ccaatggtgc caccatcgtg gc#atctttcc   1680tgtaagtcta acatatcaca aacacatcat caactccaaa cttacaaatc tc#tttatagt   1740ggctcaaaga ctggcatcgg tggctacgtc gcgaccgact ctgcacgcaa gg#aaatcgtc   1800ctctcggttc gcggtagcac caacattcgc aactggctta ccaacctcga ct#tcgaccag   1860gatgactgca gcttgacctc cggctgtgga gtgcacggag gcttccagag ag#cctggaat   1920gagatctcgg ccgcagcaac cgccgctgtc gcaaaggccc gcaaggcgaa cc#cttcgttc   1980aaggtcgttg ccacaggtca ctcgttgggt ggtgctgtag ctacactagc ag#gtgcgaac   2040ctgcgagttg gtggtacgcc agttgacatc tacacctacg gctcaccccg ag#ttggaaac   2100acgcaactcg ctgccttcat ctctaaccag gctggtggag agttccgcgt ta#cgaacgcc   2160aaggaccccg tgcctcgtct cccccctctg gtctttggat accggcacac at#cccccgag   2220tactggttgt ctggtagcgg aggtaacaag gttgactaca ccatcaatga tg#tcaaggtg   2280tgtgagggtg ctgccaacct tcagtgcaac ggtggaacac tcggattgga ta#tcgacgcc   2340catctccact acttccagga gaccgatgct tgctctggtt ccggtatcgc gt#ggagaaga   2400tacaggagtg ctaagcgtga gagcatctcg gagagggcca ctatgacaga tg#ccgagctg   2460gagaagaagc ttaacaacta tgttgcgatg gataaggagt atgtcaagac tc#acgccaac   2520cgctcatcgt agtatgacat ttacgcagta acgatataat taccataaca aa#aactctgg   2580ataccattct ggtgcaagca tggcgaagaa aacatcatta tctatgtgaa tg#tatcataa   2640ccatccttac gccatgccgt tgatcttact actgagacaa aatactcagt ca#tgtacaac   2700aaactccaaa gcaccgaatg acttctggct ttttggcaaa gcacgaaacc aa#tcattcaa   2760acccctccac gaccatgccc tgcgcattgg gaacacccac gagaatgaca cc#acgaggca   2820cgcggacact cttcaccttc atgcacccaa agacattgac ttcccggata tt#agggcatg   2880ctcggaaaat ggaacccaga acaaaatccg tcactgcctc acagaaactg at#ctccaatt   2940 <210> SEQ ID NO 2 <211> LENGTH: 349 <212> TYPE: PRT<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 2Met His Leu Leu Ser Leu Leu Ser Ile Ala Th #r Leu Ala Val Ala Ser 1               5   #                10   #                15Pro Leu Ser Val Glu Asp Tyr Ala Lys Ala Le #u Asp Glu Arg Ala Val            20       #            25       #            30Ser Val Ser Thr Asn Asp Phe Gly Asn Phe Ly #s Phe Tyr Ile Gln His        35           #        40           #        45Gly Ala Ala Ala Tyr Cys Asn Ser Glu Ala Al #a Ala Gly Ala Lys Val    50               #    55               #    60Thr Cys Gly Gly Asn Gly Cys Pro Thr Val Gl #n Ser Asn Gly Ala Thr65                   #70                   #75                   #80Ile Val Ala Ser Phe Leu Gly Ser Lys Thr Gl #y Ile Gly Gly Tyr Val                85   #                90   #                95Ala Thr Asp Ser Ala Arg Lys Glu Ile Val Le #u Ser Val Arg Gly Ser            100       #           105       #           110Thr Asn Ile Arg Asn Trp Leu Thr Asn Leu As #p Phe Asp Gln Asp Asp        115           #       120           #       125Cys Ser Leu Thr Ser Gly Cys Gly Val His Gl #y Gly Phe Gln Arg Ala    130               #   135               #   140Trp Asn Glu Ile Ser Ala Ala Ala Thr Ala Al #a Val Ala Lys Ala Arg145                 1 #50                 1 #55                 1 #60Lys Ala Asn Pro Ser Phe Lys Val Val Ala Th #r Gly His Ser Leu Gly                165   #               170   #               175Gly Ala Val Ala Thr Leu Ala Gly Ala Asn Le #u Arg Val Gly Gly Thr            180       #           185       #           190Pro Val Asp Ile Tyr Thr Tyr Gly Ser Pro Ar #g Val Gly Asn Thr Gln        195           #       200           #       205Leu Ala Ala Phe Ile Ser Asn Gln Ala Gly Gl #y Glu Phe Arg Val Thr    210               #   215               #   220Asn Ala Lys Asp Pro Val Pro Arg Leu Pro Pr #o Leu Val Phe Gly Tyr225                 2 #30                 2 #35                 2 #40Arg His Thr Ser Pro Glu Tyr Trp Leu Ser Gl #y Ser Gly Gly Asn Lys                245   #               250   #               255Val Asp Tyr Thr Ile Asn Asp Val Lys Val Cy #s Glu Gly Ala Ala Asn            260       #           265       #           270Leu Gln Cys Asn Gly Gly Thr Leu Gly Leu As #p Ile Asp Ala His Leu        275           #       280           #       285His Tyr Phe Gln Glu Thr Asp Ala Cys Ser Gl #y Ser Gly Ile Ala Trp    290               #   295               #   300Arg Arg Tyr Arg Ser Ala Lys Arg Glu Ser Il #e Ser Glu Arg Ala Thr305                 3 #10                 3 #15                 3 #20Met Thr Asp Ala Glu Leu Glu Lys Lys Leu As #n Asn Tyr Val Ala Met                325   #               330   #               335Asp Lys Glu Tyr Val Lys Thr His Ala Asn Ar #g Ser Ser            340       #           345 <210> SEQ ID NO 3 <211> LENGTH: 35<212> TYPE: DNA <213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 3cagtgaattg gcctcgatgg ccgcggccgc gaatt        #                  #       35 <210> SEQ ID NO 4 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 4aattcgcggc cgcggccatc gaggccaatt cactg        #                  #       35 <210> SEQ ID NO 5 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 5cacgaaggaa agacgatggc tttcacggtg tctg        #                  #        34 <210> SEQ ID NO 6 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 6cagacaccgt gaaagccatc gtctttcctt cgtg        #                  #        34 <210> SEQ ID NO 7 <211> LENGTH: 46 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 7ctatctcttc accatggtac cttaattaaa taccttgttg gaagcg   #                 46 <210> SEQ ID NO 8 <211> LENGTH: 46 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 8cgcttccaac aaggtattta attaaggtac catggtgaag agatag   #                 46 <210> SEQ ID NO 9 <211> LENGTH: 37 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 9cgttctttgt ctgtcagcat gcatctccta tcactcc       #                  #      37 <210> SEQ ID NO 10 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Fusarium venenatum <400> SEQUENCE: 10ccagagtttt tgttatggtt aattaatatc gttactgcgt aaatg    #                  #45

What is claimed is:
 1. An isolated nucleic acid sequence encoding apolypeptide having lipase activity, selected from the group consistingof: (a) a nucleic acid sequence encoding a polypeptide having an aminoacid sequence which has at least 90% identity with amino acids 31 to 349of SEQ ID NO:2; (b) a nucleic acid sequence having at least 90% homologywith nucleotides 1525 to 2530 of SEQ ID NO:1; (c) a nucleic acidsequence which hybridizes under high stringency conditions with (i)nucleotides 1525 to 2530 of SEQ ID NO:1, (ii) the cDNA sequencecontained in nucleotides 1525 to 2530 of SEQ ID NO:1, or (iii) a fullcomplementary strand of (i) or (ii); and (c) a subsequence of (a), (b),or (c), encoding a polypeptide fragment that has lipase activity.
 2. Thenucleic acid sequence of claim 1, which encodes polypeptide having anamino acid sequence which has at least 90% identity with amino acids 31to 349 of SEQ ID NO:2.
 3. The nucleic acid sequence of claim 1, whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.4. The nucleic acid sequence of claim 3, which encodes a polypeptidecomprising the amino acids 31 to 349 of SEQ ID NO:2.
 5. The nucleic acidsequence of claim 1, which encodes a polypeptide consisting of the aminoacid sequence of SEQ ID NO:2 or a fragment thereof which has lipaseactivity.
 6. The nucleic acid sequence of claim 5, which encodes apolypeptide consisting of the amino acid sequence of SEQ ID NO:2.
 7. Thenucleic acid sequence of claim 6, which encodes a polypeptide consistingof amino acids 31 to 349 of SEQ ID NO:2.
 8. The nucleic acid sequence ofclaim 1, which has at least 90% homology with nucleotides 1525 to 2530of SEQ ID NO:1.
 9. The nucleic acid sequence of claim 1, whichhybridizes under high stringency conditions with (i) nucleotides 1525 to2530 of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides1525 to 2530 of SEQ ID NO:1, or (iii) a full complementary strand of (i)or (ii).
 10. The nucleic acid sequence of claim 1, which has the nucleicacid sequence of SEQ ID NO:1.
 11. The nucleic acid sequence of claim 1,which is contained in plasmid pEJG60 which is contained in E. coil NRRLB-30333.
 12. A nucleic acid construct comprising the nucleic acidsequence of claim 1 operably linked to one or more control sequenceswhich direct the production of the polypeptide in a suitable expressionhost.
 13. A recombinant expression vector comprising the nucleic acidconstruct of claim 12, a promoter, and transcriptional and translationalstop signals.
 14. A recombinant host cell comprising the nucleic acidconstruct of claim
 12. 15. A method for producing a polypeptide havinglipase activity comprising (a) cultivating the host cell of claim 14under conditions suitable for production of the polypeptide; and (b)recovering the polypeptide.